Method for identifying BPDE-added target protein

Abstract

The invention relates to the field of identification of BPDE-added target proteins, in particular to a method for identifying a BPDE-added target protein. The method comprises the steps of: 1) cell collection and protein extraction; 2) contamination and protease digestion of cell lysates; 3) mass spectrum pre-sample preparation; 4) LC-MS / MS analysis: taking peptide fragments to perform chromatographic separation, and performing DDA mass spectrometry by using a mass spectrometer after peptide fragment separation; and 5) mass spectrum database search: downloading a protein database, and obtaining a BPDE target protein according to mass spectrum analysis data. By adopting the method, the target protein which can be added with BPDE in the protein lysates can be directly identified, no chemicalmodification needs to be performed on micromolecules in a detection process, the original activity of the micromolecules is not affected, no influence is generated by any cell or tissue type, no requirement is proposed for pure protein, and even whole cell lysates can be used, so that the method can be widely applied to the identification of micromolecule-added target proteins.

Description

technical field [0001] The invention relates to the identification field of BPDE adducting target protein, in particular to an identification method of BPDE adducting target protein. Background technique [0002] Benzo[a]pyrene (B[a]P) is the most common environmental and occupational pollutant. Recent studies have shown that neurological abnormalities such as cognitive impairment, learning difficulties, parasympathetic disorders and short-term memory loss are all related to B[a]P exposure. Rats exposed to B[a]P showed impaired short-term learning and spatial memory and weakened long-term potentiation (LTP) in the Morris water maze test; population epidemiological survey results showed that B[a]P [a]P occupationally exposed coke oven workers showed reduced learning and memory performance with a dose-response relationship with B[a]P exposure levels. However, the molecular mechanism of B[a]P neurotoxicity remains unclear. B[a]P is converted in vivo to the highly mutagenic a...

AI Technical Summary

Problems solved by technology

[0004] 1. For the adducted target protein of BPDE, it is usually speculated through the change of content or function, and there is no direct evidence

[0005] 2. The identification method of other small molecular compound adducted target protein needs to chemically modify the small molecular compound, such as adding biological labels such as biotin or fluorescein. The modification process may change the activity of the small molecule

Compared with traditional methods, the adducted target protein screening technology based on this principle does not require any modification and labeling of small molecules, preserves the original characteristics of binding to the target protein and is independent of any biological effects of the drug, avoiding the need for any modification of the small molecule. Therefore, it is especially suitable for the target recognition of drugs with complex structures like BPDE that cannot be chemically modified. However, in the method, the protein after protease digestion needs to be passed through After separation by SDS-PAGE electrophoresis, the differential bands were cut out and identified by in-gel enzymatic hydrolysis of the differential bands. Only a few individual adducted target proteins could be identified, which greatly reduced the information of the identified proteins.

Therefore, for the purpose of identifying BPDE adducting target proteins, there is currently a lack of a method that does not require chemical modification of small molecules and can identify BPDE adducting target proteins in large quantities.

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